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Plant Cell Physiol, 39(5): 516-525 (1998)
JSPP © 1998
Plasma Membrane Permeability of Root-Tip Cells Following Temporary
Exposure to Al Ions Is a Rapid Measure of Al Tolerance among Plant
Species
Satoru Ishikawa 1 ' 2 and Tadao Wagatsuma1'3
1
2
Laboratory of Plant Nutrition and Soil Science, Faculty of Agriculture, Yamagata University, Wakaba 1-23, Tsuruoka, Yamagata,
997 Japan
CREST, Japan Science and Technology Corporation (JST)
No correlations were recognized between Al tolerance
among four plant species, rice (Oryza sativa L.), maize
(Zea mays L.), pea (Pisunt sativum L.), and barley (Hordeum vulgare L.), in rank order of Al tolerance, and cation
exchange capacities of root-tip (0-1 cm) cells or of their cell
walls. The plasma membrane of root-tip of Al sensitive
plant species (pea and barley) was considerably permeabilized with elongation of root in Al-free solution following
0.5 h pretreatment with Al. K+ release from and Al permeation into the protoplasts isolated from the root-tip of Alsensitive plant species were more significant than those for
Al-tolerant plant species (rice and maize) on 10 or 30 min
treatment with Al. The permeability of the plasma membrane for protoplasts isolated from Al sensitive plant
species was considerably increased by treatment with hypotonic Al-free control solution following 10 min pretreatment with Al. To our knowlege, these are the most rapid responses to Al ions reported to date, i.e., within 0.5 h in
whole plant and within 10 min in protoplast. These results
suggest that a temporary contact with Al ions irreversibly
alters the plasma membrane of root-tip cells of Al-sensitive
plant species: the cells become more leaky and rigid due to
binding of Al ions to the plasma membrane.
Key words: Aluminum (Al) tolerance — CEC of cell wall —
PM permeability — Protoplast.
Aluminum (Al) toxicity is known to be a major factor
limiting plant growth in acid soils. Micromolar concentrations of Al can inhibit root elongation of Al-sensitive
wheat cultivar within 1 h (Ownby and Popham 1989). Altolerant plant species (Wagatsuma et al. 1995a) and Altolerant wheat lines (Delhaize et al. 1993a) accumulate less
Al in the root-tip than Al-sensitive species. Several mechanisms for this reduced accumulation have been proposed, including metabolism-dependent exclusion of Al (Waga-
tsuma 1983, Zhang and Taylor 1989), and detoxification of
Al by the secretion of organic acids (Delhaize et al. 1993b,
de la Fuente et al. 1997, Ma et al 1997).
The binding of Al ions to carboxyl groups in pectic
substances of the cell wall inhibits apoplastic movement of
water and nutrients (Blarney and Dowling 1995) and the metabolism of cell wall polysaccharides (Le Van et al. 1994).
In addition, Rengel (1992) speculated that Al ions repress
the cell elongation induced by acid-mediated wall loosening. However, there is no consensus on the correlation between CEC of roots and Al tolerance among plant species
or cultivars (Munn and McCollum 1976, Mugwira and
Elgawhary 1979, Wagatsuma 1983, Rengel and Robinson
1989b, Allan et al. 1990, Masion and Bertsch 1997).
Al ions immediately inhibit not only the influx of K +
(Miyasaka et al. 1989), Ca 2+ (Rengel and Elliott 1992),
Mg 2+ (Rengel and Robinson 1989a), or NOf (Durieux et
al. 1995) by the binding of Al ions to the PM protein, but
also accelerate the efflux of K + (Wagatsuma et al. 1995a),
or phosphate ions (Ownby 1993) across the PM lipid. Chen
et al. (1991) observed a decrease in water and increase in
nonelectrolyte (for example, urea) permeation across cell
membranes in intact Quercus rubra L. root cortex cells on
Al treatment, and suggested the alteration is induced by the
binding of Al 3+ to negatively charged phospholipid head
groups in cell membranes. Vierstra and Haug (1978) using
electron paramagnetic resonance spectroscopy showed that
Al 3+ decresed membrane fluidity in isolated and intact cell
membranes of Thermoplasma acidophilum. Yamamoto et
al. (1996) suggested that Al significantly induced the peroxidation of the PM lipid of cultured tobacco (Nicotiana
tabacum L.) cells at logarithmic phase in the presence of Fe
with cell death resulting from the increase in permeability
of the PM. Zhang et al. (1997) showed several genotype-specific changes in the lipid composition of the PM from roots
of wheat cultivars that differed in tolerance to Al on 3-d
treatment with Al. Jones and Kochian (1995) firstly reported in plants that Al 3+ could inhibit the phosphoinositide signal-transduction pathway. The significance of PM
of root-tip cells in Al tolerance is still unclear.
Abbreviations: CEC, cation exchange capacity; FDA, fluoresIn the present study we suggest that the PM of the
cein diacetate; IPP, the increase in PM permeability; PI, proroot-tip for Al-tolerant plant species does not easily leak
pidium iodide; PM, plasma membrane.
3
and is less susceptible in the short-term (within 1 h) to Al.
Corresponding author.
516
PM permeability as a rapid measure of Al tolerance
Materials and Methods
Plant materials and growth conditions—Seeds of rice (Oryza
saliva L. cv. Sasanishiki; an Al-tolerant plant species), maize (Zea
mays L. cv. Pioneer 3352; a plant species that is moderately
tolerant to Al), and pea (Pisum sativum L. cv. Kinusaya; an Alsensitive plant species) were soaked in tap water composed of 8
mg liter"1 Ca 2+ , 3 mg liter"1 Mg 2+ , and 1.5 mg liter"1 K + under
aeration for 1 d. Seeds of barley (Hordeum vulgare L. cv. Manriki; a plant species that is very sensitive to Al) were not soaked,
but germinated on a nylon screen that was put on a polypropylene
container filled with 8 liters of tap water under aeration at 27°C in
a growth room under fluorescent white light (18 ftE m~2 s"1) with
a 14-h photoperiod. To maintain high humidity, each container
was covered with a polyvinyl sheet and the seeds sprayed with
deionized water. Three or four-d-old seedlings with roots approximately 5 cm in length were used in the experiments.
Value of Al tolerance—The length of the longest root was
measured for ten seedlings with a ruler prior to the treatments
with control or Al. These seedlings were then transferred to the 8liters of 0.2 mM CaCl2 solution with (Al) or without (control) 5
fiM A1C13 and treated for 3 d under aeration at 27°C. The solutions were renewed every day, and pH was maintained at 5.0 by using diluted HC1 and NaOH every 6 to 8 h. After 3 d, the root
lengths of the same ten seedlings were measured again. Al tolerance was calculated based on the ratio of the net root elongation
in Al treatment to that in control.
Cell wall isolation and determination of CEC—Cell wall was
isolated basically by the method of Whitman and Travis (1985), as
described below. Twenty g of apical 1-cm segments of fresh root
was homogenized with 100 ml of a homogenizing medium consisting of 50 mM Tris-HEPES (pH 7.8), 5 mM EGTA, 5 mM
EDTA, 10 mM NaF, 2.5 mM DTT, 100/ig ml" 1 butylated hydroxytoluene, and 250 mM sucrose with a homogenizer (HG 300;
Hitachi, Tokyo, Japan) at 15,000 rpm for 30 s three times. The homogenates were filtered through a nylon cloth of pore diameter 30
nm, and fully washed with deionized water. This procedure was
repeated twice. The crude cell wall was soaked in the homogenizing medium for 24 h at 5°C, filtered with a nylon cloth, homogenized twice in a mixture of chloroform and methanol ( 1 : 2 , v/v) for
30 s each time, and filtered. The homogenates were soaked in the
mixture for 24 h at 5°C, filtered and finally washed with deionized
water. The final material on the nylon cloth was designated as purified cell wall. The purity of this material was judged by its phosphorus (P) concentration. P was determined colorimetrically by
the molybdeum blue method after digestion with a mixture of concentrated HNO3 and 60% HC1O4 ( 5 : 3 , v/v).
The CECs of apical 1 cm root segments and purified cell wall
material were determined according to the method of Wagatsuma
(1983). Briefly, dry powder (0.2 g) of root and cell wall was incubated first in 40 ml of 0.5 M HC1 for 3 h, then in 80 ml of 0.1 M
Ca(CH3COO)2 containing 0.2 mM CaCl2 (pH 5.0) for 3h and
washed with deionized water until Cl" free. The adsorbed Ca was
desorbed by incubating with 50 ml of 0.05 M HC1, and the Ca concentration in the filtrates was determined by inductively coupled
plasma atomic emission spectroscopy (ICP; Liberty 220, Varian
Australia Pty., Ltd., Victoria, Australia).
Permeability of the PM and length of re-elongation of root—
Seedlings of four plant species were treated with a 0.2 mM CaCl2
solution containing 0 (control), 5, 20, or 100 j/M A1C13 (Al) for 0.5
(rice, barley and maize) or 1 h (pea). The pH of each Al solution
and its control solution were equalized with the corresponding
values when 10 mM A1CI3 stock solution was diluted in each Al
517
concentration with deionized water; 5.0 for 5^M Al, 4.9 for 20
/iM Al, and 4.6 for 100//M Al. Permeability of roots was observed by staining with fluorescein diacetate-propidium iodide
(FDA-PI) according to the method of Ishikawa et al. (1996).
Briefly, a stock solution of FDA (Aldrich Chemical Company,
Inc., Milwaukee, WI, U.S.A.) or PI (Sigma, St. Louis, MO,
U.S.A.) was prepared by dissolving 5 mg ml" 1 in acetone or 40,ug
ml" 1 in deionized water, respectively. Roots were stained for 10
min with a mixture of FDA (12.5 j/g ml" 1 )-?! (5/igml" 1 ) solutions, both diluted with deionized water. The concentration of PI
was 15^gml - 1 for pea roots. After extra dye was removed by
washing with deionized water for 1 min, the root-tip was observed
under a stereoscopic microscope (SMZ-10; Nikon, Tokyo, Japan)
equipped with a macro-fluorescence apparatus (MX-100F; Nikon)
(excitation filter, 450-490 nm; barrier filter, 520 nm), and photographed using a photographic apparatus (AFX-II; Nikon) and a
film of ASA 400. Experiments were replicated more than three
times.
Roots which had been pretreated with the 0.2 mM CaCl2 solution containing 0, 5, 20, or 100 /iM A1C13 for 0.5 or 1 h were washed with deionized water for a few seconds, and thereafter treated
with a solution of 0.2 mM CaCl2 with or without 2 mM citrate
(pH 4.5) for 0.5 h at 5°C. The roots were washed with deionized
water, and re-elongated in 0.2 mM CaCl2 (pH 4.9) for 5 (rice, barley and maize) or 8 h (pea) at 27°C. The permeability of the re-elongated roots was observed by staining with FDA-PI as described
above. Pea roots of each of the 10 seedlings were pretreated with
0.2 mM CaCl2 solution containing 0, 5, 20, or 100 ^M A1C13 for 1
h, then treated with or without 2 mM citrate (pH 4.5) for 0.5 h at
5°C, and finally re-elongated in 0.2 mM CaCl2 (pH 4.9) for 24 h.
The net lengths of re-elongation were compared.
Permeability of protoplasts isolated from root-tip—The isolation and purification of protoplasts from root-tip were carried out
as described by Wagatsuma et al. (1995b). Briefly, apical 1 cm segments were cut into 2 mm pieces and segments weighing 3 g were
digested by shaking at 30 cycles min" 1 for 3.5 h at 27°C with 20
ml of a medium composed of 0.6 M mannitol, 2% (w/v) Cellulase
Onozuka RS (Yakult Pharmaceutical Industry Co., Ltd., Tokyo,
Japan), 0.1% (w/v) Pectolyase Y-23 (Seishin Pharmaceutical Industry Co., Ltd., Tokyo, Japan), 0.05% (w/v) BSA (fraction V;
Sigma), 1 mM CaCl2, and 0.5 mM DTT (pH 5.6). Protoplasts
were separated from the digested tissues by passage through a
nylon cloth with a pore diameter of 95 fim. The residue on the
nylon cloth was gently agitated in suspension medium, which consisted of 0.7 M mannitol, 0.1 mM CaCl2, 0.5 mM DTT, and 5 mM
Tris-MES (pH 6.5), and then filtered through the nylon cloth.
Both flitrates were centrifuged at 200 x g for 6 min and the pellet
of crude protoplasts was gently suspended in 30% (w/v) Ficoll
(Ficoll 400; Pharmacia Biotech, Uppsala, Sweden) in 0.7 M mannitol and 2 mM Tris-MES (pH 6.5). A discontinuous gradient was
formed by successive layering of solutions of 8, 5, and 0% Ficoll
in 0.7 M mannitol and 2 mM Tris-MES (pH 6.5). The fraction at
the interface between the layers of 0% and 5% Ficoll was collected
after centrifugation at 380 xg and the Ficoll was removed by
washing twice with 0.7 M mannitol solution. Protoplasts, in
which the cell wall was hardly discernible by staining with fluorescent brightener 28 (Sigma), could be isolated by this procedure
(data not shown).
Four ml of 0.2 mM CaCl2 in 0.7 M mannitol (pH 4.5,
isotonic control solution) was added to the purified protoplasts
which were then incubated for 10 min at room temperature. This
suspension was centrifuged at 130 x-g for 5 min and the supernatant was discarded. Next, 4 ml of isotonic control solution was
518
PM permeability as a rapid measure of Al tolerance
added and the protoplast pellet resuspended. The suspension was
divided into 4 aliquots (of approximately 1 ml). Three ml of the
isotonic control solution was added to two of these 1-ml aliquots
(pH 4.5, isotonic control pretreatment), and 1 ml of isotonic control solution and 2 ml of 0.2 mM CaCl2 containing 200 #M A1C13
in 0.7 M mannitol were added to the other two aliquots (pH 4.5,
isotonic Al pretreatment); the Al concentration in the latter solution was 100 fiM. These suspensions were incubated for 10 min at
27°C, then centrifuged at 130 x g for 5 min, and the supernatants
were discarded. Two ml each of 0.2 mM CaCl2 in 0.5 M mannitol
(pH 4.5, hypotonic treatment) or 0.2 mM CaCl2 in 0.7 M mannitol (pH4.5, isotonic treatment) was added to each pellet, and
the mixtures were incubated for 5 min at 27°C. After centrifugation at 130 x g for 5 min, each supernatant was reduced to 0.5 ml
by pipetting out. A few drops of the solution of FDA-PI (FDA,
25 fi% ml" 1 ; PI, 8 fig ml" 1 ) were added to the residues. The preparations were observed under a fluorescence microscope equipped
with B2 filter (excitation filter, 450-490 nm; barrier filter, 520 nm)
(EFDA-2; Nikon), and photographed as described above. The increase in PM permeability (IPP) was estimated from the ratio of
the number of protoplasts exhibiting red fluorescence to the total
number of protoplasts. Experiments were replicated more than
three times.
K+ release from protoplasts—Protoplasts isolated from roottip were incubated with isotonic control solution for 10 min, then
centrifuged at 200 x g for 5 min. Four ml of isotonic control solution was added to the pellet of protoplasts and mixed. The suspension was divided into two portions and the protoplasts were
enumerated with a hemacytometer (Erma Inc., Tokyo, Japan).
Two ml of isotonic control (0.2 mM Ca) or isotonic Al (0.2 mM
Ca containing 200 //M Al) solution was added to each portion and
the mixtures were treated for 0.5 h at 27°C. The protoplast suspensions were centrifuged at 300 x g for 5 min and the K concentration of the supernatants was determined by atomic absorption
spectrophotometry (170-50 A; Hitachi).
Al concentration of protoplasts and of protoplast ghosts—
Two ml of each solution (pH 4.5), i.e., isotonic control solution
and 0.2 mM Ca without mannitol, was added to each protoplast
pellet of which the number of protoplasts had been counted, and
incubated for 10 min. Two ml of isotonic 0.2 mM CaCl2 plus 200
fiM A1C13 solution was added to the former and 2 ml of 0.2 mM
CaCl2 plus 200^M A1C13 solution without mannitol to the latter.
These mixtures were treated for 10 min at 27°C. Mixtures were
filtered through a cellulose acetate membrane filter (pore size, 0.1
nm). The protoplasts on the membrane filters were washed several times with deionized water. The membrane filter materials were
soaked in 10 ml of 0.1 M HC1 for 24 h to extract Al. Al was determined by ICP.
Results
Relationships between Al tolerance and CEC of roots
or cell wall—Fig. 1 shows the correlations between Al tolerance among the four plant species and CECs of the roottips (0-1 cm) or of purified cell wall isolated from root-tips.
When treated with 5fiM Al for 3 d , the plants ranked as
follows in terms of Al tolerance; rice (79.9%) > maize
(69.2%) > pea (59.6%)>barley (4.4%). The CEC of roottip of rice, maize, and barley, which are monocotyledons,
was approximately 10cmol c (kg dry weight)"' of root,
whereas that of pea, a dicotyledon, was approximately 2
times that value. The CEC of the purified cell wall of monocotylendons was approximately 10 cmolc (kg dry weight)"1
of cell wall and that of pea approximately 6 times higher.
The P concentration of the purified cell wall as a percentage of that of the root-tip for each plant species was 3 to
5% (data not shown); more than 95% of the protoplasm
was removed from the cell wall materials. The correlation
coefficients between Al tolerance and CEC of root-tip or of
cell wall from root-tip among the four species was 0.011
and 0.109, respectively, and each correlation was non-significant.
Changes in permeability of the PM of root-tip cells
and in root elongation following temporary exposure to
Al ions—When stained with FDA-PI, cells with normal
permeablity can exclude PI from their PMs; in such cells,
FDA passes through PM and is hydrolyzed by intracellular
esterases to produce fluorescein, and exhibits green fluorescence when excited by UV light. On the other hand, the permeabilized cells exhibit a bright red fluorescence by the
passage of PI through their PMs and intercalation with
DNA and RNA. When the seedlings were treated with control solution above pH4.5, the PM permeability of roottip cells was normal in all plant species (data not shown).
Just after the roots of rice and barley were treated with 100
liM Al for 0.5 h, normal permeability of the PM was apparently retained in the root-tip cells (Fig. 2A, E). Normal
permeability of the PM was also demonstrated in maize (da-
0
20
40
60
80
Al tolerance (%)
100
Fig. 1 Correlation between Al tolerance and CEC of root-tip
(0-1 cm) or cell wall isolated from root-tip among four plant species. Al tolerance (%) was calculated based on the ratio of net root
elongation of the longest root of plant treated with 5 /iM Al for 3
d to that of control. The purity of cell wall isolated by homogenizing with homogenizing medium and subsequently chloroformmethanol was approximately 95% in every plant species based on
the comparison of P concentrations between root-tip and cell
wall. CEC was determined as the amount of Ca saturation to cation exchange sites at pH 5. The correlation coefficients between
Al tolerance and CEC of root-tip (o) and cell wall (A) were 0.011
and 0.109, respectively (not significant). The experiment was duplicated and values are represented with SE.
PM permeability as a rapid measure of Al tolerance
519
Fig. 2 Effect of a short Al treatment and subsequent root re-elongation on permeability of the PM in root-tip. Seedlings were pretreated with 5, 20, or 100//M A1C13 for 0.5 h, and the roots were re-elongated in Al-free 0.2 mM CaCl2 solution for 5 h. The re-elongated
roots were stained with FDA-PI. Green fluorescence and red fluorescence exhibit normality or increase of permeability of the PM, respectively. A-D and E-H, root-tip portions of barley and rice, respectively; A and E, just after the treatment with 100 fiM A1C13 for 0.5 h; B
and F, C and G, D and H, after re-elongation of roots pretreated with 5, 20, or 100//M Al, respectively. Scale bar indicates 1 mm.
520
PM permeability as a rapid measure of Al tolerance
Fig. 3 Effect of the treatment of citrate on permeability of the PM in root-tip when re-elongated following a short pretreatment with
Al. Pea seedlings were pretreated with 5, 20, and 100 fiM Al for 1 h, and the Al-pretreated roots were treated with or without 2 mM
citrate (pH 4.5) for 0.5 h, and subsequently, re-elongated in Al-free solution for 8 h. A, just after the treatment with 100//M Al; B-G,
after the re-elongation of roots following the pretreatment with 5 (B and E), 20 (C and F), or 100 (D and G) fiM Al and the treatment
with (E-G) or without (B-D) 2 mM citrate. Scale bar indicates 1 mm.
PM permeability as a rapid measure of Al tolerance
ta not shown) and pea (Fig. 3A). When the roots of Al-pretreated plants were re-elongated for 5 to 8 h in Al-free 0.2
mM CaCl2 solution, the permeability of root-tip cells, especially in the epidermis and the outer cortex cells in the elongation zone of barley (Fig. 2B-D) and pea (Fig. 3B-D), increased significantly with increasing concentrations of Al
pretreatment. Permeability of root-tip cells of maize (data
not shown) and rice (Fig. 2F-H) re-elongated in the same
way was normally retained irrespective of the concentrations used in Al pretreatment.
In pea roots which had been pretreated with 5, 20, or
100 ^M A1C13 for 1 h and treated with 2mM citrate (pH
4.5) for 0.5 h and thereafter re-elongated in Al-free 0.2 mM
CaCl2 solution for 8 h, the permeability of the PM of roottip cells was retained irrespective of the concentrations in
Al pretreatment (Fig. 3E-G). Similar results were observed
for other plant species (data not shown). The permeability
of control roots without Al pretreatment was normal at all
pH and temperature settings in the present experimental
conditions (data not shown).
The elongation of pea root in Al-free 0.2 mM CaCl2 solution for 24 h following 1 h-pretreatment with different
concentrations of Al was inhibited proportionally to the
concentrations in Al treatment, i.e., approximately 10 and
20% inhibitions at 20 and 100//M A1C13, respectively
(Fig. 4). Treatment with citrate following 1 h-pretreatment
with Al abrogated the inhibition of root elongation.
Effect of temporary exposure to Al ions on permeability of the PM of, K+ release from, and Al uptake by the
protoplasts isolated from root-tip—The permeability of
the PM for pea protoplasts treated with isotonic control
solution for 10 min was almost normally retained as
these protoplasts exhibited green fluorescence (16% of IPP,
Fig. 5A). By the 10 min-treatment with isotonic 100/iM
AICI3 solution, pea protoplasts exhibited slightly more red
fluorescence, which means a slight increase in PM permeability (30% of IPP, Fig. 5B). After the 5 min-treatment
with hypotonic control solution, the permeability of the
PM was almost normally retained in pea protoplasts pretreated with the isotonic control solution (20% of IPP,
D
•
120
521
-Citrate
+ Citrate
100
80
60
CD
40
JO
20
0
5 uM
20 uM
Al concentrations in pretreatment
Fig. 4 Effect of the treatment of citrate on the re-elongation of
pea roots pretreated temporarily with Al ions. Pretreatment with
Al ions and treatment with citrate were carried out as described
for Fig. 3. Roots were re-elongated in 0.2 mM CaCl2 solution without Al for 24 h. Relative net root re-elongation was calculated based on the ratio of the re-elongation of roots pretreated with Al to
that of roots pretreated without Al. The columns and the vertical
bars in the figure are the mean and the SE in ten seedlings.
Fig.5C), whereas it was considerably increased in pea
protoplasts pretreated with the isotonic IOOJUM A1C13 solution (66% of IPP, Fig. 5D). Considerably less permeabilization was observed in rice protoplasts on the same treatment
( < 2 5 % o f IPP, Fig.5E-H).
Based on the relative K+ release from protoplasts treated with isotonic 100//M A1C13 solution for 0.5 h to that
from protoplasts treated with isotonic control solution, the
plants ranked in the order; barley, p e a > > maize> rice
(Table 1).
For Al content of protoplasts treated with 100 /uM
AICI3 for 10 min, the plants ranked as follows: barley > maize > pea > rice (Table 2). Little amount of the cell wall
material was presumed to be the surface of protoplasts be-
Table 1 Effect of Al ions on K+ release from protoplasts
Rice
Maize
Pea
Barley
Control
0.36±0.07
0.95 ±0.02
Al
0.34±0.03
1.02±0.03
0.94
1.07
0.27±0.03
0.51 ±0.07
1.89
0.64±0.08
1.21 ±0.03
1.89
Al/Control
100
Protoplasts were treated with isotonic control (Control) or 100/iM Al (Al) solutions for 0.5 h. After
centrifugation, the supernatants were collected and the K + concentration in the supernatants was determined using atomic absorption spectrophotometry. The values of Control and Al are means ±SE
in duplicate and represented as [pmol K+ (protoplast)"']. The value of Al/Control indicates the ratio
of K + release in both treatments.
522
PM permeability as a rapid measure of Al tolerance
Fig. 5 Effect of Al ions and hypotonic conditions on permeability of the PM of protoplasts. Protoplasts isolated from root-tip of pea
(A-D) and rice (E-H) were pretreated with isotonic control (A and E) or 100//M Al (B and F) solution for 10 min, and subsequently
treated with hypotonic Al-free solution for 5 min. Protoplasts were treated with hypotonic treatment following pretreatment with
isotonic control (C and G) and Al solution (D and H). Protoplasts were observed under a fluorescence microscope following staining
with FDA-PI. Scale bar indicates 100 ftm.
PM permeability as a rapid measure of Al tolerance
523
Table 2 Al contents of protoplasts and protoplast ghosts
Protoplasts (A)
Protoplast ghosts (B)
(B)/(A)
Rice
Maize
Pea
Barley
20 .9±4 .3
59 .9±4 .2
58 .7±2.0
104 .3±9.2
48 .6±5 .9
57 .9±1 .7
62 .3±3. 2
74 .4±7. 8
2.87
1.78
1.19
1.19
(A), protoplasts isolated from root tip (0-1 cm) were treated with isotonic 100 /JM Al solution for 10
min; (B), protoplast ghosts were prepared by the treatment of protoplasts with control solution in the
absence of 0.7 M mannitol, and subsequently with 100 fiM Al solution in the absence of 0.7 M mannitolfor 10 min. The values of (A) and (B) are means (n=3) ±SE of Al contents of protoplasts [fmol
Al (protoplast)"1] and protoplast ghosts [fmol Al (protoplast ghost)" 1 ], respectively. The value of
(B)/(A) indicates the ratio of Al content of protoplast ghosts to that of protoplasts.
cause of the short period of the treatment (within 30 min)
and the simple composition of the nutrient solution (Mock
et al. 1990). Protoplast ghosts took up more Al in all plant
species, but on the ratio of the Al content of protoplasts to
that of protoplast ghosts the plants ranked as follows:
rice >maize> pea, barley (Table 2).
Discussion
Most investigations on mechanisms of Al tolerance
have treated cultivars or near-isogenic lines differing in Al
tolerance within the same species. In the present study, we
focused on the effect of Al ions on the permeability of PM
and the negativities of cell wall and PM among several plant
species that differ widely in Al tolerance.
Munn and McCollum (1976) and Wagatsuma (1983)
reported no correlation between root CEC and Al tolerance, whereas Mugwira and Elgawhary (1979) and Rengel
and Robinson (1989b) suggested a negative correlation between the two. In the present experiment, no correlation
was recognized between root CEC and Al tolerance among
the four plant species (r=0.011, Fig. 1). We also found no
correlation between the two among ten plant species and
cultivars within the same species that differ in Al tolerance
(data not shown). The CEC of the purified cell wall from
pea was approximately 6 times greater than those from the
other species and was approximately 2.6 times greater than
the root CEC of pea (Fig. 1). The CECs of cell walls from
the other species were similar to those of roots. The high
value of the CEC of cell wall from pea may be attributable
to a considerable removal of cellular components with little
negative charge in the preparation of cell wall materials. A
strong positive correlation was recognized between root
CEC and the CEC of cell wall among the four plant species
(r=0.994, data not shown). Although EDTA and EGTA
used in cell wall isolation may partly remove water soluble
and loosely bound pectins in cell wall, the difference in the
CECs of cell walls among the species was consistent with
the difference in pectin contents among them (Sakurai et al.
1991). No correlation was recognized between cell wall
CEC and Al tolerance among the species (r = 0.109, Fig. 1).
Cosgrove and co-workers found two proteins (expansins)
involved in wall extension from the cell wall of growing
hypocotyls, and reported that Al 3+ inhibited both their activities (McQueen-Mason et al. 1992, Cosgrove 1996). However, rapid response to a temporary contact with Al ions
(within 1 h) is assumed to be characteristic of the PM of
root-tip cells (Fig. 2, 3, 4). Short-term contact with Al ions
(10 min) revealed a similar effect on the PM of cell-wall
removed cells, i.e., protoplast (Fig. 5). These results suggest a reduced significance of cell wall materials in Al tolerance. The role of expansins in Al tolerance remains to be
elucidated.
Ishikawa et al. (1996) suggested an association of permeability of the PM with Al tolerance on treatment with
20 fiM A1C13 for 2 h. In the present experiment, temporary
treatment with Al ions (less than 1 h) did not induce any
change in permeability of the PM and no relation between
permeability and Al tolerance was observed (Fig. 2A, E,
3A). However, when roots pretreated with Al ions for a
short period (less than 1 h) were re-elongated in Al-free solution, the PM of root-tip cells was clearly permeabilized
depending on the Al concentrations in treatment and on Al
tolerance (Fig.2B-D, F-H, 3B-D). These results suggest
that the PM of Al-sensitive plant species becomes rigid and
less extensible on short-term contact with Al ions. The PM
of protoplasts isolated from Al-sensitive plant species,
which had been pretreated with isotonic Al solution for 10
min, was permeabilized considerably after the 5 min-treatment with hypotonic Al-free control solution (66% of IPP,
Fig. 5D). These results can be interpreted as follows: in Alsensitive plant species, contact with Al ions induces a rigid
PM, reduces the extension of it, permeabilizes it during the
re-elongating period or under hypotonic conditions, and inhibits root elongation.
When Al-pretreated roots were treated with citrate for
0.5 h, and subsequently re-elongated in Al-free solution for
several hours, the permeability of the PM in root-tip cells
524
PM permeability as a rapid measure of Al tolerance
was retained in every plant species irrespective of the Al
concentrations in pretreatment (Fig. 3E-G). The 0.5 h
of treatment with 2 mM citrate at pH 4.5 results in the desorption of the apoplastic Al bound to the cell wall and
outer leaflet of PM (Ownby and Popham 1989, Zhang
and Taylor 1989), though some Al ions enter the symplast
(Lazof et al. 1994). In addition to this, the permeation of
citric acid and aluminum citrate through the lipid bilayer is
limited by hydrogen bonding between hydrated water molecules and carboxyl groups of citrate, and the hydrated
water shell of Al ions (Akeson and Munns 1989). The recovery of normal permeability of the PM by treatment of
citrate is ascribable to the desorption of Al from the PM.
Al associated with protoplast is composed of two fractions: Al bound to the PM and permeated Al. Microtubule,
nucleus and cytoplasmic proteins may be retained in the
protoplast ghost (Sonobe and Takahashi 1994), and therefore the permeated Al ions can easily bind to symplastic
contents. A greater Al content in protoplast ghost relative
to protoplast indicates difficulty in permeation of protoplast by Al ions. Table 2 suggests that the PM of root-tip
for Al-tolerant plant species is less permeable to Al ions.
The smaller release of K + from protoplast (Table 1) and
root-tip portion (Wagatsuma et al. 1995a) of Al-tolerant
plant species supports this. That there were no differences
in K + release from (Table 1) and Al permeation into (Table
2) the protoplasts between pea and barley in spite of the significant difference in Al tolerance between them (Fig. 1)
may be due to the high concentration of Al in the medium,
i.e., 100yuM Al.
Ishikawa et al. (1996) investigated the comparative
toxicity of Al 3+ , Yb 3 + , and La 3+ to root-tip cells and suggested that Al 3+ with the largest ionic potential among the
three metal ions most preferentially binds covalently to
the negative sites of PM. When hydrated Al 3+ , i.e., [Al
(OH 2 ) 6 ] 3+ , binds to the negatively charged phospholipid,
seven to eight hydrated water molecules of the phosphate
group (Cevc 1982) and six hydrated water molecules of
Al 3+ may be dehydrated together by the Eigen mechanism.
Dispersed phospholipid molecules in the normal PM form
a partial packing area by binding Al ions; membrane in a
liquid crystal state becomes rigid and gel-like as a result of
dehydration (Hauser and Phillips 1979, Chen et al. 1991).
This dehydration may make the packing area more hydrophobic. Membrane proteins can also join this packing area
with their carboxyl groups and Al ions. The boundary between the packed area (e.g., phospholipids and proteins)
and non-packed area (e.g., sterols) may be enlarged by reelongation or under hypotonic conditions; permeabilization of the PM may be the result of the notch effect of
stress concentration in a mechanical sense.
We speculated that the negativity of the PM of roottip cells determines the extent to which Al ions bind to the
PM of root-tip cells and finally the Al tolerance of several
plant species (Wagatsuma and Akiba 1989, Wagatsuma et
al. 1995a, b). Takabatake and Shimmen (1997) and Jones
and Kochian (1997) reported phospholipids or lipids of PM
as the primary site for Al toxicity. Yermiyahu et al. (1997)
suggested that PM surface negativity and Al sorptive capacity probably account for some of the sensitivity to Al 3+ . On
the other hand, Zhang et al. (1997) reported that the ratio
of phospholipids to total lipid in the PM from whole roots
was smaller in Al-sensitive wheat cultivar. Further investigations on the characteristics of PM in association with Al
tolerance should be carried out. Al tolerance among plant
species is rapidly revealed in the intactness of the PM permeability of root-tip cells after temporary exposure to Al
ions. Similar results have been observed between cultivars
of several plant species in other experiments, and this will
be reported elsewhere. To the authors' knowledge, no investigations have been reported on this close association
following short-term Al treatment; less than 0.5 h in whole
plant and 10 min in protoplast.
Whether or not the amount of organic acids exuded
from root-tip is enough to detoxify Al ions in medium is
still unclear, though there is a higher concentration of exuded organic acids near the surface and in the cell wall of
root. We have explained one of the mechanisms of Al tolerance even in protoplasts free from such apoplastic space.
We thank Dr. H. Ikarashi (Experimental Farm of Yamagata
University) for supplying rice seeds.
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